Stem cells having increased sensitivity to a chemoattractant and methods of generating and using same

ABSTRACT

The present invention relates to stem cells which exhibit increased sensitivity to a chemoattractant and, more particularly, to methods of generating and using them such as in clinical applications involving stem cell transplantation.

This application is a U.S. National Phase Application pursuant to 35U.S.C. 371 of International Application No. PCT/IL2004/000315, which wasfiled Apr. 7, 2004, claiming benefit of priority of Israel PatentApplication No. 155303, which was filed Apr. 8, 2003, and Israel PatentApplication No. 159307, which was filed Dec. 10, 2003. The entiredisclosure of each of the foregoing applications is incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to stem cells which exhibit increasedsensitivity to a chemoattractant and, more particularly, to methods ofgenerating and using them such as in clinical applications involvingstem cell transplantation.

BACKGROUND OF THE INVENTION

Medical treatment of disorders caused by abnormal organ functiontypically employ pharmaceutical agents designed for either compensatingfor such abnormal organ function or treating the dysfunctional organtissue. However, in some cases, pharmaceutical therapy cannot beinstated since organ function is oftentimes complex and/or notcompletely understood.

In such cases, the only viable alternative is surgical replacement ofthe non-functional organ, which is now widely used for treatment ofliver and kidney failure, both acute and chronic, as well as for cancerand certain inborn abnormalities. However, the need for donor organs farexceeds the supply. Organ shortage has resulted in new surgicaltechniques, such as splitting adult organs for transplant. Despitefairly good results, such techniques still suffer from a lack of donortissue.

The lack of viable donor tissue has led to the emergence of stem cellreplacement therapy, which relies on stem cell plasticity i.e., theability to give rise to cell types in a new location that are notnormally present in the organ in which the stem cells are located.

Stem cells are generally classified according to their origin,essentially adult, embryonic or neonatal origin. Embryonic stem cellsderiving from the inner cell mass of the blastocyst are pluripotential,bring capable of giving rise to cells found in all three germ layers.Despite long held belief adult stem cells are not as lineage restrictedas previously thought. In particular, haematopoietic and neural stemcells appear to be the most versatile at cutting across lineageboundaries. For example, recent reports suggest that hematopoietic stemcells (HSCs) of human origin have a hepatic potential. Studies of liveror bone marrow transplantation from sex mismatched donors, identifiedbone marrow-derived hepatocytes in recipients [Alison (2000) Nature406:257; Theise (2000) Hepatology 32:11-16; Korbling (2002) N Engl J Med346:738-746]. Murine and rat HSCs were also found to migrate toirradiated or injured adult livers, and to differentiate into hepaticcells [Petersen (1999) Science 284:1168-1170; Theise (2000) Hepatology31:235-240; Lagasse (2000) Nat Med 6:1229-1234]. Furthermore, singlemurine hematopoietic stem cell transplantation has resulted in detectionof HSC-derived cells in the liver of irradiated recipients with a lowpercentage of transplanted cells exhibiting immunohistochemical andmorphologic properties of hepatic epithelial cells [Krause (2001) Cell105:369-377].

The mechanisms that guide circulating hematopoietic stem cells areclinically significant because the success of stem cell transplantationdepends on efficient targeting (also referred to as homing) of graftedcells to the recipient target tissue [Mazo and von Adrian (1999) Journalof leukocyte Biology 66,25-32]. It is due to this homing of transplantedcells that bone marrow transplantations do not require invasive surgery,as in the case with the transplantation of any other organ, but rathercan be effected by simple intravenous infusion.

Homing of HSCs can be defined as the set of molecular interactions thatallows circulating HSCs to recognize, adhere to, and migrate across bonemarrow endothelial cells resulting in the accumulation of HSCs in theunique hematopoiesis-promoting microenvironment of the bone marrow.Homing of progenitor cells can be conceived as a multi-step phenomenon[Voermans (2001) J. Hematother. Stem Cell Res. 10:725-738, Lapidot(2002) Leukemia 16:1992-2003]. HSCs arriving to the bone marrow mustfirst interact with the luminal surface of the bone marrow endothelium.This interaction must occur within seconds after the HSCs have enteredthe bone marrow microvasculature and provide sufficient mechanicalstrength to permit the adherent cell to withstand the shear forceexerted by the flowing blood. Adherent HSCs must then pass through theendothelial layer to enter the hematopoietic compartment. Afterextravasation, HSCs encounter specialized stromal cells whosejuxtaposition supports maintenance of the immature pool by self-renewalprocess in addition to lineage-specific HSCs differentiation,proliferation and maturation, a process that involves stroma-derivedcytokines and other growth signals.

Only a limited number of factors involved in stem cells homing are knownto date; these include, the ligand for c-kit, stem cell factor, whichhas been shown to play a central role in adherence of HSCs to the stroma[Peled (1999) Science 283:845-848]; and integrin interactions (e.g.,β1-Intergrins), which were shown to be crucial to the migration of HSCsto the foetal liver [Zanjani (1999) Blood 94:2515-2522].

One important molecular interaction which is considered central to HSChoming is that of chemokine stromal derived factor (SDF-1) and itscognate receptor, CXCR4.

SDF-1 is the only known powerful chemoattractant of hematopoietic stemcells of both human [Aiuti (1997) J. Exp. Med. 185:111-120] and murineorigin [Wright (2002) J. Exp. Med. 195:1145-1154] known to date. SDF-1is widely expressed in many tissues during development [McGrath (1999)Dev. Biol. 213:442-456] and adulthood [Nagasawa (1994) Proc Natl AcadSci USA 91:2305-2309; Imai (1999) Br J Haematol 106:905-911; Pablos(1999) Am J Pathol 155:1577-1586], such as for example the liver[Shirozu (1995) Genomics 28:495-500; Nagasawa (1996) Nature 382:635-638;Goddard (2001) Transplantation 72:1957-1967]. Previously, the presentinventors were able to show that bone marrow homing and repopulation bysorted human CD34⁺/CD38^(−/low) stem cells transplanted into the tailvein of irradiated immune deficient NOD/SCID and NOD/SCID/B2m null mice,are dependent on SDF-1/CXCR4 interactions [Peled (1999) Science283:845-848; Kollet (2001) Blood 97:3283-3291]. More recently, thepresent inventors also established a role for these interactions inG-CSF-induced mobilization of murine and human stem cells [Petit (2002)Nat Immunol 3:687-694].

In view of the ever-expanding use of stem cell therapy, it is highlydesirable to further elucidate the mechanism behind stem cell homing andtarget repopulation in order to improve the efficiency and success rateof cell replacement therapy.

Hepatocyte growth factor (HGF), initially identified as a potent mitogenfor mature hepatocytes, is a kringle-containing polypeptide growthfactor sharing structural homology with plasminogen [Nakamure (1984)Biohcem. Biophys. Res. Commun. 122:1450; Nakamura (1987) FEBS Lett.224:311; Gohda (1988) J. Clin. Invest. 81:414; Zanegar Cancer Res.49:3314; Nakamura (1989) Nature 342:440].

HGF is a mesenchyme-derived pleiotropic factor which regulates cellgrowth, cell motility and morphogenesis in various types of cells[Matsumoto (1993) Goldberg I D, Rosen E M (eds): Hepatocyte GrowthFactor-Scatter Factor (HG-SF) and C-met Receptor, Basel, Switzerland,Birkhauser Verlag, 1993, 225; Gherardi (1990) Nature 346:228; Weidner(1990) J. Cell Biol. 111:2097; Higasho (1990) Biochem. Biophys. Res.Commun. 170:397; Rubin (1991) Proc. Natl. Acad. Sci. USA 88:415]. C-metproto-oncogene is the natural and only receptor for HGF known to date.HGF is considered a humoral mediator of epithelial-mesenchymalinteractions responsible for organogenesis of various tissues andorgans, regeneration of organs and growth, invasion and metastasis oftumor cells [Matsumoto (1996) J. Biochem. 119:591]. In the hematopoieticsystem, HGF augments the growth of hematopoietic progenitor cells[Kmiecik (1992) Blood 80:2454; Nishino (1995) Blood 85:3093; Mizuno(1993) Biochem Biophys Res. Commun. 194:178 Galimi (1994) J. Cell Biol.127:1743). Interestingly, an acute liver injury has been reported totrigger expression of HGF as determined by in-situ hybridization of HGFafter stimulation of rat liver with carbon tetrachloride [CCl4, Armbrust(2002) Liver 22:486-494].

Information pertaining to the interaction of HGF with hematopoieticcells is incomplete, although a possible role in hematopoiesis has beensuggested. HGF was found to be constitutively produced by human bonemarrow stromal cells [Takai (1997) Blood 89:1560-1565]. Recently, HGFwas found to be synergistic with GM-CSF and IL-3 in proliferation ofmurine myeloid progenitor cell line and murine hemopoietic progenitorcells (HPCs) enriched from bone marrow (BM) or fetal liver [Kmiecik(1992) Blood 80:2454-2457; Mizuno (1993) Supra; Nishino (1995) Blood85:3093-3100]. In addition, a synergistic proliferative effect of HGFwith other growth factors on human HPCs has been observed and expressionof C-met on CD34+ HPC was detected as well [Galami (1994) J. Cell Biol.127:1743-1754; Goff (1996) Stem Cells 14:592-602; Weimer (1998) Exp.Hematol. 26:885-894].

While reducing the present invention to practice the present inventorshave uncovered that HGF can upregulate CXCR4 expression and promoteSDF-1/CXCR4 dependent stem cell motility and migration to the targettissue. These findings provide a novel approach for sensitizing stemcell recruitment to a target tissue and as such can be used in variouscell and tissue replacement protocols.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of increasing sensitivity of stem cells to a chemoattractant, themethod comprises exposing the stem cells to HGF or an active portionthereof, which is capable of increasing a level of at least onechemoattractant receptor of the stem cells to thereby increase thesensitivity of the stem cells to the chemoattractant.

According to another aspect of the present invention there is provided amethod of treating a disorder requiring cell or tissue replacement, themethod comprises providing to a subject in need thereof atherapeutically effective amount of stem cells pretreated with HGF or anactive portion thereof, which is capable of increasing a level of atleast one chemoattractant receptor of the stem cells, thereby treatingthe disorder requiring cell or tissue replacement in the subject.

According to yet another aspect of the present invention there isprovided a method of treating a disorder requiring cell or tissuereplacement, the method comprises providing to a subject in need thereofa therapeutic effective amount of HGF or an active portion thereof,which is capable of increasing a level of at least one chemoattractantreceptor of stem cells, thereby treating the disorder requiring cell ortissue replacement.

According to still another aspect of the present invention there isprovided a use of HGF or an active portion thereof for the manufactureof a medicament for increasing homing of stem cells to a target tissue.

According to an additional aspect of the present invention there isprovided a method of generating stem cells suitable for transplantation,the method comprises: (a) collecting stem cells; (b) exposing the stemcells to HGF or an active portion thereof; and (c) isolating stem cellshaving CXCR4 levels above a predetermined threshold, to thereby generatestem cells suitable for transplantation.

According to further features in preferred embodiments of the inventiondescribed below, collecting the stem cells is effected by: (i) a stemcell mobilization procedure; and/or (ii) a surgical procedure.

According to still further features in the described preferredembodiments the isolating stem cells having CXCR4 levels above thepredetermined threshold is effected by FACS.

According to still further features in the described preferredembodiments the method further comprises determining homing capabilitiesof the stem cells having CXCR4 levels above the predetermined thresholdfollowing step (c).

According to yet an additional aspect of the present invention there isprovided a nucleic acid construct comprising a first polynucleotidesequence encoding HGF or an active portion thereof and an induciblecis-acting regulatory element for directing expression of thepolynucleotide in cells.

According to still further features in the described preferredembodiments the inducible cis-acting regulatory element is a shearstress activation element.

According to still further features in the described preferredembodiments the nucleic acid construct further comprises a secondpolynucleotide sequence being translationally fused to the firstpolynucleotide sequence, the second polynucleotide sequence encoding asignal peptide capable of directing secretion of the HGF or the activeportion thereof out of the cells.

According to still an additional aspect of the present invention thereis provided a eukaryotic cell comprising the nucleic acid construct.

According to a further aspect of the present invention there is provideda cell-line comprising stem cells transformed to express an exogenouspolynucleotide encoding HGF or an active portion thereof.

According to yet a further aspect of the present invention there isprovided a cell culture comprising: (i) stem cells; and (ii) feedercells expressing HGF or an active portion thereof each being capable ofincreasing a level of at least one chemoattractant receptor of the stemcells.

According to still a further aspect of the present invention there isprovided a method of increasing sensitivity of stem cells to achemoattractant, the method comprises, upregulating an expression oractivity of endogenous HGF or an active portion thereof of the stemcells to thereby increase the sensitivity of the stem cells to thechemoattractant.

According to still a further aspect of the present invention there isprovided a method of increasing stem cell motility, the method comprisesexposing the stem cells to HGF or an active portion thereof which iscapable of increasing motility of the stem cells.

According to still an additional aspect of the present invention the atleast one chemoattractant receptor is CXCR4.

According to still an additional aspect of the present invention themethod further comprises exposing the stem cells to a growth factorand/or a cytokine.

According to still an additional aspect of the present invention thegrowth factor and/or cytokine are selected from the group consisting ofSCF and IL-6.

According to still an additional aspect of the present invention thestem cells are hematopoietic stem cells.

According to still an additional aspect of the present invention thehematopoietic stem cells are CD34⁺ hematopoietic stem cells.

According to still an additional aspect of the present invention thehematopoietic stem cells are CD34⁺/CD38^(−/low) hematopoietic stemcells.

According to still an additional aspect of the present invention thestem cells are mesenchymal stem cells.

According to still an additional aspect of the present inventionexposing the stem cells to the HGF or the active portion thereof, iseffected by: (i) expressing a polynucleotide encoding the HGF or theactive portion thereof in the stem cells; and/or (ii) contacting thestem cells with the HGF or the active portion thereof.

According to still an additional aspect of the present invention themethod further comprises exposing the stem cells to HGF-receptor.

According to still an additional aspect of the present inventionprovides a method of facilitating self repopulation and/or selfengraftment of self progenitor cells to an injured organ, comprisingadministration of HGF or an active portion thereof alone or togetherwith SCF to a subject suffering of organ inflammation and/or injury.According to still an additional aspect of the present inventionprovides a pharmaceutical composition comprising HGF or an activeportion thereof which is capable of increasing motility of the stemcells and SCF, and more specifically to a pharmaceutical composition fortreating a disorder requiring cell or tissue replacement.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing stem cells, which exhibitincreased sensitivity to a chemoattractant and methods of generating andusing the same.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

plurality of instructions.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 a is a graph depicting cytokine induced expression of CXCR4 in CBCD34⁺ cells as determined using flow cytometry.

FIG. 1 b is a histogram depicting SDF-1 mediated directional migrationof CD34⁺ cells in the presence of HGF, SCF or a combination thereof, asdetermined using a Transwell migration assay. Data represent percentageof migration. The (a) character denotes spontaneous migration in theabsence of SDF-1.

FIG. 2 Shows increased expression of HGF mRNA in the BM and liver ofirradiated mice. NOD/SCID mice were sublethally irradiated (375 cGy).Liver and BM samples collected 24 and 48 hours later, together with asample from a non-irradiated mouse, were homogenized in Tryreagent(MRC). MRNA was extracted using standard protocol. RNA was subjected toRT-PCR.

FIG. 3 Shows that HGF increases the potential of BM cells to migratetowards SDF-1 and the rate of progenitor cell mobilization, followingCCl4 injury. NOD/SCID mice were treated with a single injection of CCl4alone (10 ml, CCl4), or with CCl4 followed by 4 consecutive dailyinjections of HGF (1.5 mg/mouse, CCl4+HGF) starting 2 days later.SDF-1-induced migration by BM cells and the level of progenitors in theblood circulation of these mice were compared to non treated mice (ctrl)or to mice treated with G-SCF (300 mg/Kg, 5 consecutive days).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to stem cells which exhibit increasedsensitivity to a chemoattractant and to methods of generating and usingthe same. Specifically, the present invention allows to treat disordersrequiring cell or tissue replacement such as for example to treatchronic or acute liver damage.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

The use of cellular therapy is growing rapidly, and is graduallybecoming an important therapeutic modality in treatment of variousdisorders. Hematopoietic stem cell (HSC) (e.g., bone marrow, umbilicalcord blood or mobilized peripheral blood) transplantation is one exampleof a routinely practiced, insurance-reimbursed cellular therapy.However, many other cellular therapies are being developed as well,including immunotherapy for cancer and infectious diseases, chondrocytetherapy for cartilage defects, neuronal cell therapy forneurodegenerative diseases, and stem cell therapy for numerousapplications [Forbes (2002) Clinical Science 103:355-369].

One of the problems associated with stem cell therapy is the difficultyof achieving long-term successful engraftment of cells at the targettissue. Currently, patients which were successfully transplanted exhibitvery low levels of stem cells and immature progenitors which generatecells with the desired phenotype.

Thus, the success of stem cell transplantation depends on the ability ofintravenously infused stem cells to lodge in the target tissue (e.g.,bone marrow), a process referred to as homing. It is hypothesized thathoming is a multistep process, consisting of adhesion of the stem cellsto endothelial cells of the marrow sinusoids, followed bytransendothelial migration directed by chemoattractants, and finallyanchoring within the extravascular bone marrow spaces whereproliferation and differentiation will occur.

Studies have shown that numerous factors are involved in the homingprocess including, adhesion molecules, cytokines and growth factors. In1997 studies uncovered that migration of CD34⁺ cells was goverened bythe chemoattractant, SDF-1. Subsequent studies have shown that SDF-1activates integrins on HSCs and induces trans-endothelial migration ofHSCs in vitro. The receptor for SDF-1 is a G-protein coupled receptor,termed CXCR-4. In SDF-1 or CXCR-4 knock-out mice hematopoieticprecursors do not shift to the bone marrow during fetal developmentsuggesting that SDF-1/CXCR4 interactions play an important role in stemcell migration [for review see Voermans (2001) J. Hematother. Stem CellRes. 10:725-738, Lapidot (2002) Leukemia 16:1992-2003].

Despite preliminary understanding of the homing process, informationabout regulation of migration of stem cells is still incomplete andscattered. It is well appreciated that improving the efficacy of stemcell transplantation may be achieved by modulating the ability of stemcells to home to the target tissue.

While reducing the present invention to practice the present inventorshave uncovered that HGF upregulates CXCR4 expression and promotesSDF-1/CXCR4 dependent stem cell motility and migration to a damagedtarget tissue.

As illustrated hereinunder and in the Examples section which follows,the present inventors illustrate that hepatic injury upregulates HGF,which induces cytoskeletal rearrangements, increases the motility andpotentiates the response of immature CD34+ cells to SDF-1 signaling byinducing CXCR4 upregulation and synergizing with stem cell factor (SCF).

Although HGF has been previously shown to be upregulated following liverinjury [Armbrust Liver 2002 December; 22(6):486-94], the presentinventors are the first to show that this upregulation in HGF activityleads to upregulation in CXCR4 expression to cytoskeletal rearrangementsand accelerated homing of cells expressing the same.

The present findings enable the generation of stem cells, which can beefficiently recruited to a target tissue and as such can be used innumerous clinical applications, such as in repair of liver injury and inliver or bone marrow transplantation.

Thus, according to one aspect of the present invention there is provideda method of increasing sensitivity of stem cells to a chemoattractant.

According to another aspect of the present invention there is provided amethod comprising the administration of HGF to a subject suffering oforgan inflammation and/or injury, to facilitate self repopulation and/orself engraftinent to the injured organ, due to increased progenitor stemcell levels in the cell blood circulation.

As used herein, the phrase “stem cells” refers to cells, which arecapable of differentiating into other cell types having a particular,specialized function (i.e., “fully differentiated” cells).

The method according to this aspect of the present invention includesexposing the stem cells to HGF or an active portion thereof which iscapable of increasing the level of at least one chemoattractant receptorof the stem cells to thereby increase the sensitivity of the stem cellsto the chemoattractant.

Alternatively, increasing sensitivity of stem cells to a chemoattractantcan also be effected by upregulating expression or activity ofendogenous HGF of the stem cells.

As is further described herein under, exposing the stem cells to HGF oran active portion thereof can be effected by either contacting the cellswith the protein or an active portion thereof, or by expressing theprotein or an active portion thereof within these cells or expressingHGF or an active portion thereof in non-stem cells cultured therewith(e.g., fibroblasts used as a feeder layer).

As is clearly demonstrated in the Examples section which follows,exposure of stem cells to HGF substantially increased their motility andtheir ability to migrate to a chemoattractant thereof i.e., SDF-1.

The invention relates to HGF and to its salts, functional derivatives,precursors and active fractions as well as its active mutants, i.e.other proteins or polypeptides wherein one or more amino acids of thestructure are eliminated or substituted by other amino acids or one ormore amino acids were added to that sequence in order to obtainpolypeptides or proteins having the same activity of the HGF andcomprises also the corresponding “fusion proteins” i.e. polypeptidescomprising the HGF or a mutation thereof fused with another protein. TheHGF can therefore be fused with another protein such as, for example, animmunoglobulin.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of the HGF protein of the inventionor muteins thereof. Salts of a carboxyl group may be formed by meansknown in the art and include inorganic salts, for example, sodium,calcium, ammonium, ferric or zinc salts, and the like, and salts withorganic bases as those formed, for example, with amines, such astriethanolamine, arginine or lysine, piperidine, procaine and the like.Acid addition salts include, for example, salts with mineral acids suchas, for example, hydrochloric acid or sulphuric acid, and salts withorganic acids such as, for example, acetic acid or oxalic acid. Ofcourse, any such salts must have substantially similar activity to theHGF protein of the invention or its muteins.

The definition “functional derivatives” as herein used refers toderivatives which can be prepared from the functional groups present onthe lateral chains of the amino acid moieties or on the terminal N- orC-groups according to known methods and are comprised in the inventionwhen they are pharmaceutically acceptable i.e. when they do not destroythe protein activity or do not impart toxicity to the pharmaceuticalcompositions containing them. Such derivatives include for exampleesters or aliphatic amides of the carboxyl-groups and N-acyl derivativesof free amino groups or O-acyl derivatives of free hydroxyl-groups andare formed with acyl-groups as for example alcanoyl- or aroyl-groups.

“Fragment” of the protein the present invention refers to any fragmentor precursor of the polypeptidic chain of the compound itself, alone orin combination with related molecules or residues bound to it, forexample residues of sugars or phosphates, or aggregates of thepolypeptide molecule when such fragments or precursors show the sameactivity of the HGF as medicament.

The term “circularly permuted” as used herein refers to a linearmolecule in which the termini have been joined together, either directlyor through a linker, to produce a circular molecule, and then thecircular molecule is opened at another location to produce a new linearmolecule with termini different from the termini in the originalmolecule. Circular permutations include those molecules whose structureis equivalent to a molecule that has been circularized and then opened.Thus, a circularly permuted molecule may be synthesized de novo as alinear molecule and never go through a circularization and opening step.The particular circular permutation of a molecule is designated bybrackets containing the amino acid residues between which the peptidebond is eliminated. Circularly permuted molecules, which may includeDNA, RNA and protein, are single-chain molecules which have their normaltermini fused, often with a linker, and contain new termini at anotherposition. See Goldenberg, et al. J. Mol. Biol., 165: 407-413 (1983) andPan et al. Gene 125: 111-114 (1993), both incorporated by referenceherein. Circular permutation is functionally equivalent to taking astraight-chain molecule, fusing the ends to form a circular molecule,and then cutting the circular molecule at a different location to form anew straight chain molecule with different termini. Circular permutationthus has the effect of essentially preserving the sequence and identityof the amino acids of a protein while generating new termini atdifferent locations.

The terms “polypeptide and protein” in the present specification areinterchangeable.

The present invention also concerns muteins of the above HGF protein ofthe invention, which muteins retain essentially the same biologicalactivity of the HGF protein having essentially only the naturallyoccurring sequences of the HGF. Such “muteins” may be ones in which upto about 20 and 10 amino acid residues may be deleted, added orsubstituted by others in the HGF protein respectively, such thatmodifications of this kind do not substantially change the biologicalactivity of the protein mutein with respect to the protein itself.

These muteins are prepared by known synthesis and/or by site-directedmutagenesis techniques, or any other known technique suitable thereof.

Any such mutein preferably has a sequence of amino acids sufficientlyduplicative of that of the basic the HGF such as to have substantiallysimilar activity thereto. Thus, it can be determined whether any givenmutein has substantially the same activity as the basic protein of theinvention by means of routine experimentation comprising subjecting sucha mutein to the biological activity tests set forth in Examples below.

Muteins of the HGF protein which can be used in accordance with thepresent invention, or nucleic acid coding thereof, include a finite setof substantially the HGF corresponding sequences as substitutionpeptides or polynucleotides which can be routinely obtained by one ofordinary skill in the art, without undue experimentation, based on theteachings and guidance presented herein. For a detailed description ofprotein chemistry and structure, see Schulz, G. E. et al., Principles ofProtein Structure, Springer-Verlag, New York, 1978; and Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co.,San Francisco, 1983, which are hereby incorporated by reference. For apresentation of nucleotide sequence substitutions, such as codonpreferences, see. See Ausubel et al., Current Protocols in MolecularBiology, Greene Publications and Wiley Interscience, New York, N.Y.,1987-1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.

Preferred changes for muteins in accordance with the present inventionare what are known as “conservative” substitutions. Conservative aminoacid substitutions of those in the protein having essentially thenaturally-occurring HGF sequences, may include synonymous amino acidswithin a group, which have sufficiently similar physicochemicalproperties that substitution between members of the group will preservethe biological function of the molecule, see Grantham, Science, Vol.185, pp. 862-864 (1974). It is clear that insertions and deletions ofamino acids may also be made in the above-defined sequence withoutaltering its function, particularly if the insertions or deletions onlyinvolve a few amino acids, e.g., under 50, and preferably under 20 HGFand do not remove or displace amino acids which are critical to afunctional conformation, e.g., cysteine residues, Anfinsen, “PrinciplesThat Govern The Folding of Protein Chains”, Science, Vol. 181, pp.223-230 (1973). Muteins produced by such deletions and/or insertionscome within the purview of the present invention.

Preferably, the synonymous amino acid groups are those defined in TableA. More preferably, the synonymous amino acid groups are those definedin Table B; and most preferably the synonymous amino acid groups arethose defined in Table C.

TABLE A Preferred Groups of Synonymous Amino Acids Amino Acid SynonymousGroup Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, His Leu Ile, Phe,Tyr, Met, Val, Leu Pro Gly, Ala, Thr, Pro Thr Pro, Ser. Ala, Gly, His,Gln, Thr Ala Gly, Thr, Pro, Ala Val Met, Tyr, Phe, Ile, Leu, Val GlyAla, Thr, Pro, Ser. Gly Ile Met, Tyr, Phe, Val, Leu, Ile Phe Trp, Met,Tyr, Ile, Val, Leu, Phe Tyr Trp, Met, Phe, Ile, Val, Leu, Tyr Cys Ser,Thr, Cys His Glu, Lys, Gln, Thr, Arg, His Gln Glu, Lys, Asn, His, Thr,Arg, Gln Asn Gln, Asp, Set, Asn Lys Glu, Gln, His, Arg, Lys Asp Glu,Asn, Asp Glu Asp, Lys, Asn, Gln, His, Arg, Glu Met Phe, Ile, Val, Leu,Met Trp Trp

TABLE B More Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Sers Sers Arc His, Lys, Arg Leu Ile, Phe, Met, Leu ProAla, Pro Thr Thr Ala Pro, Ala Val Met, Ile, Val Gly Gly Ilea Ile, Met,Phe, Val, Leu Phe Met, Tyr, Ile, Leu, Phe Try Phi, Try Cys Ser, Cys HisArg, Gln, His Gln Glu, His, Gln Asn Asp, Asn Lys Arg, Lys Asp Asn, AspGlu FLN, Glu Met Phe, Ile, Val, Leu, Met Trp Trp

TABLE C Most Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Sers Sers Arc Arc Leu Ile, Met, Leu Pro Pro Thr TharAlan Alan Val Val Gly Gly Ilea Ile, Met, Leu Phi Phi Try Tyr Cys Ser,Cys His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Met Ile, Leu, MetTrp Trp

Examples of production of amino acid substitutions in proteins which canbe used for obtaining muteins of the protein for use in the presentinvention include any known method steps, such as presented in U.S. REPat. No. 33,653, U.S. Pat. Nos. 4,959,314, 4,588,585 and 4,737,462, toMark et al; U.S. Pat. No. 5,116,943 to Koths et al., U.S. Pat. No.4,965,195 to Namen et al; U.S. Pat. No. 4,879,111 to Chong et al; andU.S. Pat. No. 5,017,691 to Lee et al; and lysine substituted proteinspresented in U.S. Pat. No. 4,904,584 (Straw et al).

In another preferred embodiment of the present invention, any mutein ofthe HGF protein for use in the present invention has an amino acidsequence essentially corresponding to that of the above noted HGFprotein of the invention. The term “essentially corresponding to” isintended to comprehend muteins with minor changes to the sequence of thebasic protein which do not affect the basic characteristics thereof,particularly insofar as its ability to the HGF is concerned. The type ofchanges which are generally considered to fall within the “essentiallycorresponding to” language are those which would result fromconventional mutagenesis techniques of the DNA encoding the HGF proteinof the invention, resulting in a few minor modifications, and screeningfor the desired activity for example increasing the sensitivity of stemcells to a chemoattractant.

The present invention also encompasses HGF variants. A preferred HGFvariant are the ones having at least 80% amino acid identity, a morepreferred the HGF variant is one having at least 90% identity and a mostpreferred variant is one having at least 95% identity to HGF amino acidsequence.

The term “sequence identity” as used herein means that the amino acidsequences are compared by alignment according to Hanks and Quinn (1991)with a refinement of low homology regions using the Clustal-X program,which is the Windows interface for the ClustalW multiple sequencealignment program (Thompson et al., 1994). The Clustal-X program isavailable over the Internet atftp://ftp-igbmc.u-strasbg.fr/pub/clustalx/. Of course, it should beunderstood that if this link becomes inactive, those of ordinary skillin the art could find versions of this program at other links usingstandard Internet search techniques without undue experimentation.Unless otherwise specified, the most recent version of any programreferred herein, as of the effective filing date of the presentapplication, is the one, which is used in order to practice the presentinvention.

Another method for determining “sequence identity” is he following. Thesequences are aligned using Version 9 of the Genetic Computing Group'sGDAP (global alignment program), using the default (BLOSUM62) matrix(values −4 to +11) with a gap open penalty of −12 (for the first null ofa gap) and a gap extension penalty of −4 (per each additionalconsecutive null in the gap). After alignment, percentage identity iscalculated by expressing the number of matches as a percentage of thenumber of amino acids in the claimed sequence.

Muteins in accordance with the present invention include those encodedby a nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNAunder stringent conditions and which encodes a the HGF protein inaccordance with the present invention, comprising essentially all of thenaturally-occurring sequences encoding the HGF and sequences which maydiffer in its nucleotide sequence from the naturally-derived nucleotidesequence by virtue of the degeneracy of the genetic code, i.e., asomewhat different nucleic acid sequence may still code for the sameamino acid sequence, due to this degeneracy.

The term “hybridization” as used herein shall include any process bywhich a strand of nucleic acid joins with complementary strand through abase pairing (Coombs J, 1994, Dictionary of Biotechnology, stoktonPress, New York N.Y.). “Amplification” is defined as the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction technologies well known in the art(Dieffenbach and Dveksler, 1995, PCR Primer, a Laboratory Manual, ColdSpring Harbor Press, Plainview N.Y.).

“Stringency” typically occurs in a range from about Tm−5° C. (5° C.below the melting temperature of the probe) to about 20° C. to 25° C.below Tm.

The term “stringent conditions” refers to hybridization and subsequentwashing conditions, which those of ordinary skill in the artconventionally refer to as “stringent”. See Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publications and WileyInterscience, New York, N.Y., 1987-1995; Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989.

As used herein, stringency conditions are a function of the temperatureused in the hybridization experiment, the molarity of the monovalentcations and the percentage of formamide in the hybridization solution.To determine the degree of stringency involved with any given set ofconditions, one first uses the equation of Meinkoth et al. (1984) fordetermining the stability of hybrids of 100% identity expressed asmelting temperature Tm of the DNA-DNA hybrid:Tm=81.5C+16.6(Log M)+0.41(% GC)−0.61(% form)−500/L

where M is the molarity of monovalent cations, % GC is the percentage ofG and C nucleotides in the DNA, % form is the percentage of formamide inthe hybridization solution, and L is the length of the hybrid in basepairs. For each 1 C that the Tm is reduced from that calculated for a100% identity hybrid, the amount of mismatch permitted is increased byabout 1%. Thus, if the Tm used for any given hybridization experiment atthe specified salt and formamide concentrations is 10 C below the Tmcalculated for a 100% hybrid according to the equation of Meinkoth,hybridization will occur even if there is up to about 10% mismatch.

As used herein, “highly stringent conditions” are those which provide aTm which is not more than 10 C below the Tm that would exist for aperfect duplex with the target sequence, either as calculated by theabove formula or as actually measured. “Moderately stringent conditions”are those, which provide a Tm, which is not more than 20 C below the Tmthat would exist for a perfect duplex with the target sequence, eitheras calculated by the above formula or as actually measured. Withoutlimitation, examples of highly stringent (5-10 C below the calculated ormeasured Tm of the hybrid) and moderately stringent (15-20 C below thecalculated or measured Tm of the hybrid) conditions use a wash solutionof 2×SSC (standard saline citrate) and 0.5% SDS (sodium dodecyl sulfate)at the appropriate temperature below the calculated Tm of the hybrid.The ultimate stringency of the conditions is primarily due to thewashing conditions, particularly if the hybridization conditions usedare those, which allow less stable hybrids to form along with stablehybrids. The wash conditions at higher stringency then remove the lessstable hybrids. A common hybridization condition that can be used withthe highly stringent to moderately stringent wash conditions describedabove is hybridization in a solution of 6×SSC (or 6×SSPE (standardsaline-phosphate-EDTA), 5×Denhardt's reagent 0.5% SDS, 100 &micro; g/mldenatured, fragmented salmon sperm DNA at a temperature approximately 20to 25 C below the Tm. If mixed probes are used, it is preferable to usetetramethyl ammonium chloride (TMAC) instead of SSC (Ausubel, 1987,1999).

Non-limiting examples of stem cells, which can be used according to thisaspect of the present invention, are hematopoietic stem cells (HSCs) andmesenchymal stem cells (MSCs) obtained from bone marrow tissue of anindividual at any age or from cord blood of a newborn individual,embryonic stem (ES) cells obtained from the embryonic tissue formedafter gestation (e.g., blastocyst), or embryonic germ (EG) cellsobtained from the genital tissue of a fetus any time during gestation,preferably before 10 weeks of gestation. Further description of stemcells, which can be used according to this aspect of the presentinvention is summarized hereinbelow.

HSCs—Hematopoietic stem cells (HSCs) are the formative pluripotentialblast cells found inter alia in bone marrow, fetal liver, umbilical cordblood and peripheral blood which are capable of differentiating into anyof the specific types of hematopoietic or blood cells, such aserythrocytes, lymphocytes, macrophages and megakaryocytes. Typically,within the bone marrow, HSCs reside in niches that support all therequisite factors and adhesive properties to maintain their ability andproduce an appropriate balanced output of mature progeny over the lifetime of the organism [Whetton (1999) Trends Cell Biol 9:233-238;Weissman (2000) Cell 100:157-168; Jankowska-Wieczorek (2001) Stem Cells19:99-107; Chan (2001) Br. J. Haematol. 112:541-557].

HSCs according to this aspect of the present invention are preferablyCD34⁺ cells and more preferably CD34⁺/CD38^(−/low) cells, which are amore primitive stem cell population and are therefore lesslineage-restricted and are the major long-term bone marrow repopulatingcells.

MSCs—Mesenchymal stem cells are the formative pluripotential blast cellsfound inter alia in bone marrow, blood, dermis and periosteum that arecapable of differentiating into more than one specific type ofmesenchymal or connective tissue (i.e. the tissues of the body thatsupport the specialized elements; e.g. adipose, osseous, stroma,cartilaginous, elastic and fibrous connective tissues) depending uponvarious influences from bioactive factors, such as cytokines.

Approximately, 30% of human marrow aspirate cells adhering to plasticare considered as MSCs. These cells can be expanded in vitro and theninduced to differentiate. The fact that adult MSCs can be expanded invitro and stimulated to form bone, cartilage, tendon, muscle or fatcells render them attractive for tissue engineering and gene therapystrategies. In vivo assays have been developed to assay MSC function.MSCs injected into the circulation can integrate into a number oftissues described hereinabove. Specifically, skeletal and cardiac musclecan be induced by exposure to 5-azacytidine and neuronal differentiationof rat and human MSCs in culture can be induced by exposure toβ-mercaptoethanol, DMSO or butylated hydroxyanisole [Tomita (1999)100:11247-11256; Woodbury (2000) J. Neurosci. Res. 61:364-370].Furthermore, MSC-derived cells are seen to integrate deep into brainafter peripheral injection as well as after direct injection of humanMSCs into rat brain; they migrate along pathways used during migrationof neural stem cells developmentally, become distributed widely andstart lose markers of HSC specialization [Azizi (1998) Proc. Natl. Acad.Sci. USA 95:3908-3913]. Methods for promoting mesenchymal stem andlineage-specific cell proliferation are disclosed in U.S. Pat. No.6,248,587.

Epitopes on the surface of the human mesenchymal stem cells (hMSCs) suchas SH2, SH3 and SH4 described in U.S. Pat. No. 5,486,359 can be used asreagents to screen and capture mesenchyrnal stem cell population from aheterogeneous cell population, such as exists, for example, in bonemarrow. Precursor mesenchymal stem cells which are positive for CD45 arepreferably used according to this aspect of the present invention, sincethese precursor mesenchymal stem cells can differentiate into thevarious mesenchymal lineages.

Preferred stem cells according to this aspect of the present inventionare human stem cells.

Table 1, below provides examples of adult stem cells, which can be usedto obtain the indicated phenotype in a target tissue of interest,according to this aspect of the present invention.

TABLE 1 Differentiated Stem cell phenotype Target tissue Reference Bonemarrow Oval cells, Liver Petersen (1999) Science 284: 1168-1170Hepatocytes KTLS cells Hepatocytes Liver Lagasse (2000) Nat. Med. 6:1229-1234 Bone marrow Hepatocytes Liver Alisan (2000) Nature 406: 257;Thiese (2000) Hepatology 32: 11-16 Pacreatic exocrine Hepatocytes LiverShen (2000) Nat. Cell Biol. 2: 879-887 cells Pacreas Hepatocytes LiverWang (2001) Am. J. Pathol. 158: 571-579 Bone marrow Endothelium LiverGao (2001) Lancet 357: 932-933 Bone marrow Tubular Kidney Poulsom (2001)J. Pathol. 195: 229-235 epithelium, glomeruli Bone marrow EndotheliumKidney Lagaaij (2001) Lancet 357: 33-37 Extra renal Endothelium KidneyWilliams (1969) Surg. Forum 20: 293-294 Bone marrow Myocardium HeartOrlic (2001) Nature 410: 701-704 Bone marrow Cardiomyocytes HeartJackson (2001) J. Clin Invest. and Endothelium 107: 1395-1402 Bonemarrow Type 1 Lung Krause (2001) Cell 105: 369-377 pneumocytes NeuronalMultiple Marrow Bjornson (1999) Science 283: 534-537 hematopoieticlineages Bone marrow Neurons CNS Mezey (2000) Science 290: 1779-1782Bone marrow Microglia and CNS Eglitis (1997) Proc. Natl. Acad. Sci.Astrocyes USA 94: 4080-4085 Abbreviations: SP—Side population cells;CNS—central nervous system;

As mentioned hereinabove the stem cells according to this aspect of thepresent invention are exposed to HGF or an active portion thereof.

HGF is a heterodimeric polypeptide including a 62 kDa α-subunit and a 34kDa β-subunit. The α-subunit contains an N-terminal hairpin domainincluding 27 amino acids followed by four canonical kringle domainswhich are 80-amino acid double looped structures stabilized by threeinternal disulfide bridges [Comoglio (1999) Exp. Cell. Res. 253:88-99;Comoglio (1993) Exs 65:131-65; Comoglio (1996) Genes Cells 1:347-54].The kringle domains are important for protein-protein interaction. Thefirst kringle domain contains the high affinity binding domain for theHGF-receptor, while the second kringle domain contains a low affinitybinding site to membrane associated heparan-sulfate proteoglycans. Thelow affinity interaction maintains a high concentration of HGF in thevicinity of target cells. The HGF gene encodes a single pro-HGF protein,which is cleaved to form the active heterodimeric molecule with highaffinity for the receptor.

As used herein an active portion of HGF, refers to the minimal HGFsequence, which is sufficient to increase sensitivity of the stem cellsof the present invention to the chemoattractant As used herein an activeportion of HGF, refers also to a mutein, fusion protein, functionalderivative, fragment, circularly permutated HGF and/or salt thereof.

To determine the active portion of HGF according to the presentinvention, stem cells can be contacted with an HGF segment and responseof the cells thereto can be monitored molecularly, biochemically orfunctionally (e.g., motility, homing, migration assays) using methodswhich are well known to those of skill in the art and are furtherdescribed hereinbelow.

As described hereinabove exposing the stem cells to HGF or an activeportion thereof increases the level of at least one type ofchemoattractant receptor of the stem cells.

A number of chemotactic cell receptors are known to participate intransendothelial migration of stem cells. Many of these receptors belongto the family of G protein-coupled seven-transmembrane receptors(7-TMR). Signaling via G proteins, particularly Gi proteins, results ina chemotactic response of the cells towards a gradient of thecorresponding ligand [Voermans (2001) J. Hematother. Stem Cell Res.10:725-738]. Recent studies have provided evidence for expression ofseveral 7-TMR on immature hematopoietic progenitor cells, whichpotentially mediate chemotactic effects: chemokine receptors (e.g.,CXCR4, receptor for stromal cell-derived factor-1), receptors for lipidmediators (e.g., the cysteinyl leukotriene receptor cysLT1 and theperipheral cannabinoid receptor cb2), and receptors for neuroendocrinehormones (e.g., the somatostatin receptor sst2). From these studies itcan be concluded that migration of hematopoietic progenitor and stemcells is controlled by a variety of chemotactic factors rather than by asingle chemokine (e.g., SDF-1).

According to preferred embodiments of this aspect of the presentinvention the chemotactic receptor is CXCR4.

It will be appreciated that since HGF exerts its biological activitiesthrough binding to an HGF-receptor i.e., c-met, the present inventionalso contemplates exposing the cells to HGF receptor, i.e. c-met whichmay be a limiting factor in HGF-signaling.

As mentioned hereinabove, exposing the stem cells to HGF or an activeportion thereof can be effected by contacting the stem cells with theprotein or by expressing the protein within the stem cells.

Contacting stem cells with HGF or an active portion thereof ispreferably effected using harvested cells, although the presentinvention also contemplates mobilization of stem cells from tissue intocirculation and exposure of circulating stem cells to the HGF or anactive portion thereof.

Adult stem cells can be obtained using a surgical procedure such as bonemarrow aspiration or can be harvested using commercial systems such asthose available from Nexell Therapeutics Inc. Irvine, Calif., USA.

Stem cells utilized by the present invention are preferably collected(i.e., harvested) using a stem cell mobilization procedure, whichutilizes chemotherapy or cytokine stimulation to release of HSCs intocirculation of subjects. Stem cells are preferably retrieved using thisprocedure since mobilization is known to yield more is HSCs andprogenitor cells than bone marrow surgery.

Stem cell mobilization can be induced by a number of molecules. Examplesinclude but are not limited to cytokines such as, granulocytecolony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interleukin (IL)-7, IL-3, IL-12,stem cell factor (SCF), and flt-3 ligand; chemolines like IL-8, Mip-1α,Groβ, or SDF-1; and the chemotherapeutic agents cyclophosphamide (Cy)and paclitaxel. It will be appreciated that these molecules differ inkinetics and efficacy, however, according to presently known embodimentsG-CSF is preferably used alone or in combination such as withcyclophosphamide to mobilize the stem cells. Typically, G-CSF isadministered daily at a dose of 5-10 μg/kg for 5-10 days. Methods ofmobilizing stem cells are disclosed in U.S. Pat. Nos. 6,447,766 and6,162,427.

Human embryonic stem cells can be isolated from human blastocysts. Humanblastocysts are typically obtained from human in vivo preimplantationembryos or from in vitro fertilized (IVF) embryos. Alternatively, asingle cell human embryo can be expanded to the blastocyst stage. Forthe isolation of human ES cells the zona pellucida is removed from theblastocyst and the inner cell mass (ICM) is isolated by immunosurgery,in which the trophectoderm cells are lysed and removed from the intactICM by gentle pipetting. The ICM is then plated in a tissue cultureflask containing the appropriate medium which enables its outgrowth.Following 9 to 15 days, the ICM derived outgrowth is dissociated intoclumps either by a mechanical dissociation or by an enzymaticdegradation and the cells are then re-plated on a fresh tissue culturemedium. Colonies demonstrating undifferentiated morphology areindividually selected by micropipette, mechanically dissociated intoclumps, and re-plated. Resulting ES cells are then routinely split every1-2 weeks. For further details on methods of preparation human ES cellssee Thomson et al., [U.S. Pat. No. 5,843,780; Science 282: 1145, 1998;Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92:7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 1989]; Gardner et al.,[Fertil. Steril. 69: 84, 1998].

It will be appreciated that commercially available stem cells can bealso be used according to this aspect of the present invention. Human EScells can be purchased from the NIH human embryonic stem cells registry(<http://escr.nih.gov>). Non-limiting examples of commercially availableembryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92,CY10, TE03, TE32.

Human EG cells can be retrieved from the primordial germ cells obtainedfrom human fetuses of about 8-11 weeks of gestation using laboratorytechniques known to anyone skilled in the arts. The genital ridges aredissociated and cut into small chunks, which are thereafterdisaggregated into cells by mechanical dissociation. The EG cells arethen grown in tissue culture flasks with the appropriate medium. Thecells are cultured with daily replacement of medium until a cellmorphology consistent with EG cells is observed, typically after 7-30days or 1-4 passages. For additional details on methods of preparing EGcells see Shamblott et al., [Proc. Natl. Acad. Sci. USA 95: 13726, 1998]and U.S. Pat. No. 6,090,622.

It will be appreciated that enrichment of stem cell populationexlubiting pluripotency may be preferably effected. Thus, for example,as outlined hereinabove, CD34⁺ stem cells can be concentrated usingaffinity columns or FACS as further described hereinunder.

Culturing of stem cells under proliferative conditions may also beeffected in cases where stem cell numbers are too low for use intreatment Culturing of stem cells is described in U.S. Pat. Nos.6,511,958, 6,436,704, 6,280,718, 6,258,597, 6,184,035, 6,132708 and5,837,5739.

Once stem cells are obtained, they are contacted with HGF or an activeportion thereof.

Soluble HGF, and in particular, active portion thereof can bebiochemically synthesized by using, for example, standard solid phasetechniques. These methods include exclusive solid phase synthesis,partial solid phase synthesis methods, fragment condensation, classicalsolution synthesis. Solid phase peptide synthesis procedures are wellknown in the art and further described by John Morrow Stewart and JanisDillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce ChemicalCompany, 1984).

Synthetic peptides can be purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing.

It will be appreciated that HGF can also be obtained from commercialsuppliers such as, Sigma-Aldrich, Rehovot, Israel; Product No. H1404.

In cases where large amounts of HGF or the active portion thereof aredesired, such polypeptides are preferably generated using recombinanttechniques.

To recombinantly synthesize such polypeptides, an expression construct(i.e., expression vector), which includes a polynucleotide encoding theHGF or the active portion thereof positioned under the transcriptionalcontrol of a regulatory element, such as a promoter, is introduced intohost cells.

The “transformed” cells are cultured under suitable conditions, whichallow the expression of the fusion protein encoded by thepolynucleotide.

Following a predetermined time period, the expressed protein isrecovered from the cell or cell culture, and purification is effected.

A variety of prokaryotic or eukaryotic cells can be used ashost-expression systems to express the modified polypeptide codingsequence. These include, but are not limited to, microorganisms, such asbacteria transformed with a recombinant bacteriophage DNA, plasmid DNAor cosmid DNA expression vector containing the desired coding sequence;Mammalian expression systems are preferably used to express the HGF orthe active portion thereof, since eukaryotic cells enable the generationof post-translational modified proteins. However, bacterial systems aretypically used to produce recombinant proteins since they enable a highproduction volume at low cost. Thus, the host system is selectedaccording to the recombinant protein to be generated and the end usethereof.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the modifiedpolypeptide expressed. For example, when large quantities of conjugatesare desired, vectors that direct the expression of high levels of theprotein product, possibly as a fusion with a hydrophobic signalsequence, which directs the expressed product into the periplasm of thebacteria or the culture medium where the protein product is readilypurified may be desired. Certain fusion protein engineered with aspecific cleavage site to aid in recovery of the conjugate may also bedesirable. Such vectors adaptable to such manipulation include, but arenot limited to, the pET series of E. coli expression vectors [Studier etal. (1990) Methods in Enzymol. 185:60-89).

Other expression systems such as insects and mammalian host cellsystems, which are well known in the art can also be used by the presentinvention (see U.S. Pat. No. 6,541,623).

In any case, transformed cells are cultured under effective conditions,which allow for the expression of high amounts of recombinantpolypeptide. Effective culture conditions include, but are not limitedto, effective media, bioreactor, temperature, pH and oxygen conditionsthat permit protein production. An effective medium refers to any mediumin which a cell is cultured to produce the recombinant modifiedpolypeptide of the present invention. Such a medium typically includesan aqueous solution having assimilable carbon, nitrogen and phosphatesources, and appropriate salts, minerals, metals and other nutrients,such as vitamins. Cells of the present invention can be cultured inconventional fermentation bioreactors, shake flasks, test tubes,microtiter dishes, and petri plates. Culturing can be carried out at atemperature, pH and oxygen content appropriate for a recombinant cell.Such culturing conditions are within the expertise of one of ordinaryskill in the art.

The resultant recombinant proteins of the present invention arepreferably secreted into the growth (e.g., fermentation) medium.

Following a predetermined time in culture, recovery of the recombinantprotein is effected. The phrase “recovering the recombinant protein”refers to collecting the whole growth medium containing the protein andneed not imply additional steps of separation or purification (seeExample 2 of the Examples section). Proteins of the present inventioncan be purified using a variety of standard protein purificationtechniques, such as, but not limited to, affinity chromatography, ionexchange chromatography, filtration, electrophoresis, hydrophobicinteraction chromatography, gel filtration chromatography, reverse phasechromatography, concanavalin A chromatography, chromatofocusing anddifferential solubilization.

Proteins of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein in thediverse applications, described hereinbelow.

It will be appreciated that recombinant production of HGF or the activeportion thereof of the present invention can also be effected in-vitro.

HGF or the active portion thereof can be included in a culture mediumutilized for culturing or sustaining the harvested stem cells. Such aculture medium typically includes a buffer solution (i.e., growthmedium) suitable for stem cell culturing. The culture medium can alsoinclude serum or serum replacement which include growth factors whichsupport growth and survival of the stem cells. The culture medium canalso include homing agents such as SDF-1, IL-6, SCF and the like.Additionally the growth medium of the present invention may also includedifferentiation-inhibiting agents such as leukemia inhibitor factor(LIF).

The stem cells of the present invention can also be contacted with HGFsecreting cells. This can be effected by co-culturing the stem cells ofthe present invention with cells which express HGF or an active portionthereof. For example, fibroblast feeder cells, which areoftentimes-co-cultured with stem cells to support proliferation thereofin a non-differentiated state, can express HGF, thereby performing adual role i.e., growth support and increase of migration and motilitypotential of stem cells.

However since the stem cells of the present invention are preferablyused for clinical applications, measures are taken to isolate the stemcells from the second HGF-expressing cell population following inductionof sufficient level of the at least one chemoattractant receptor of thestem cells. Methods of sorting cell populations are further describedhereinbelow.

Alternatively, the stem cells of the present invention can betransformed with an expression construct such as that described above inorder to express HGF or the active portion thereof in the stem cells.

In such cases, the expression construct includes a cis-acting regulatoryelement active in mammalin cells (examples above), preferably underinducible, growth specific or tissue specific conditions.

Examples of cell type-specific and/or tissue-specific promoters includepromoters such as albumin that is liver specific [Pinkert et al., (1987)Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al.,(1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cellreceptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins;[Banerji et al. (1983) Cell 33729-740], neuron-specific promoters suchas the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad.Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.(1985) Science 230:912-916] or mammary gland-specific promoters such asthe milk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). The nucleic acid construct of the presentinvention can further include an enhancer, which can be adjacent ordistant to the promoter sequence and can function in up regulating thetranscription therefrom.

Preferably, the inducible cis-acting regulatory element is regulatableby changes in the environment of the stem cells during thehoming-implantation process.

During their migration, stem cells are subjected to shear forcesgenerated by movement of the cells within circulating blood; onceimplanted, stem cells are no longer subjected to such shear forces.Since the HGF is preferably active during the homing stage (migration),the use of a cis-acting regulatory element which is active only at thestage of migration is particularly advantageous. One such regulatoryelement is the shear stress responsive element described by Resnick etal., in PNAS USA 90:4591-4595, 1993.

Genetic modification of mesenchymal stem cells is discussed in U.S. Pat.No. 5,591,625. Genetic modofocation of HSCs is discussed in Zheng 2000Nat. Biotechnol. 18:176-180 and Lotti 2002 J. Virol. 76(8)3996-4007.

Once exposed to HGF or an active portion thereof, stem cells exhibitingincreased expression levels of the chemoattractant receptor and as aresult, increased sensitivity to the chemoattractant are preferablyidentified and isolated. Although such a step enriches for highlychemotactic cells, use of a non-enriched HGF-treated population is alsoenvisaged by the present invention.

Identification and isolation of such cells according to this aspect ofthe present invention can be effected using a number of cytological,biochemical and molecular methods which are well known in the art.

For example, analysis of receptor level can be effected by flowcytometry. This approach employs instrumentation that scans single cellsflowing past excitation sources in a liquid medium. The technology canprovide rapid, quantitative, multiparameter analyses on single living(or dead) cells based on the measurement of visible and fluorescentlight emission. This basic protocol focuses on: measure fluorescenceintensity produced by fluorescent-labled antibodies and ligands thatbind specific cell-associated molecules. To isolate cell populationsusing fluorescence activated cell sorter stem cells of the presentinvention are contacted with anti CXCR4 commercially available from R&D,614 McKinley Place NE Minneapolis, Minn.

Other cytological or biochemical methods for quantitatively assessingthe level of the chemotactic receptor expression include but are notlimited to binding analysis using a labeled (e.g., radioactivelylabeled) chemokine, western blot analysis, cell-surface biotinylationand immunofluorescent staining.

It will be appreciated that the receptor expression levels can also bedetermined at the mRNA level. For example, CXCR4 mRNA may be detected incells by hybridization to a specific probe. Such probes may be clonedDNAs or fragments thereof, RNA, typically made by in-vitrotranscription, or oligonucleotide probes, usually generated by solidphase synthesis. Methods for generating and using probes suitable forspecific hybridization are well known and used in the art.Quantification of mRNA levels can be also effected using anamplification reaction [e.g., PCR, “PCR Protocols: A Guide To MethodsAnd Applications”, Academic Press, San Diego, Calif. (1990)), employingprimers, which hybridize specifically to the MRNA of a chemotacticreceptor of interest.

A variety of controls may be usefully employed to improve accuracy inmRNA detection assays. For instance, samples may be hybridized to anirrelevant probe and treated with RNAse A prior to hybridization, toassess false hybridization.

Functional assays can also be used to determine the chemotactic receptorexpression. For example, a chemotaxis assay which employs a gradient ofthe chemotactic agent (e.g., SDF-1) and follows stem cell migrationthrough a membrane towards the chemotactic agent can be utilized toidentify and isolate stem cells exhibiting increased chemotaxis. If thecells do not express enough levels of the chemotactic receptor (e.g.,CXCR4), then the majority of the cells will remain on the membrane.However, upon increased expression of the chemoattractant receptor ofthe present invention, cells will migrate through the membrane andsettle on the bottom of the well of the chemotaxis plate (see Example 1of the Examples section).

It will be appreciated that a functional homing assay can also beutilized by the method of the present invention. Such an assay isdescribed in Kollet (2001) Blood 97:3283-3291.

Immunofluorescent staining can also be employed in a functional assayfor determining cellular morphology following exposure to HGF. Asillustrated in FIG. 1 a of Example 1 which follows, co-staining withanti CXCR4 and anti-mobilized actin antibodies uncovered changes in stemcell morphology (i.e., cell spreading and lamellipodia formation)following treatment with HGF.

Stem cells exhibiting an increased sensitivity to the chemoattractantcan be used in a wide range of clinical applications.

Thus, according to another aspect of the present invention there isprovided a method of treating a disorder requiring cell or tissuereplacement. The method is effected by providing to a subject in needthereof a therapeutically effective amount of stem cells pretreated withHGF or an active portion thereof selected capable of increasing a levelof at least one chemoattractant receptor of the stem cells as describedhereinabove, to thereby treat the disorder requiring the cell or tissuereplacement in the subject.

Disorders requiring cell or tissue replacement include but are notlimited to various immunodeficiencies such as in T and/or B lymphocytes,or immune disorders, such as rheumatoid arthritis. Suchimmunodeficiencies may be the result of viral infections, HTLVI, HTLVII,HTLVIII, severe exposure to radiation, cancer therapy or the result ofother medical treatment; Hematological deficiencies including but notlimited to leukemias, such as acute lymphoblastic leukemia (ALL), acutenonlymphoblastic leukemia (ANLL), acute myelocytic leukemia (AML) orchronic myelocytic leukemia (CML). Other such hematological deficienciescan be, but are not limited to, severe combined immunodeficiency (SCID)syndromes (such as, for example adenosine deaminase (ADA) deficiency andX-linked SCID (XSCID)), osteopetrosis, aplastic anemia, Gaucher'sdisease, thalassemia and other congenital or genetically-determinedhematopoietic abnormalities; Other disorders requiring cell or tissuereplacement include those associated with liver failure, pancreticfailure, neurological disorders, those disorders requiring augmentedbone formation such as osteoartbritis, osteoporosis, traumatic orpathological conditions involving any of the connective tissues, such asa bone defects, connective tissue defects, skeletal defects or cartilagedefects.

Preferred individual subjects according to the present invention aremammals such as canines, felines, ovines, porcines, equines, bovines andpreferably humans.

The stem cells according to this aspect of the present invention arepreferably obtained from the subject to be treated. However stem cellsmay also be obtained from a syngeneic, allogeneic and less preferablyfrom a xenogeneic donor.

It will be appreciated that when allogeneic or xenogeneic stem cells areused, the recipient subject and/or cells are preferably treated toprevent graft versus host and host versus graft rejections.Immunosuppression protocols are well known in the art and some aredisclosed in U.S. Pat. No. 6,447,765.

It will be appreciated that the stem cells of the present invention canbe genetically modified to express any therapeutic gene such as anantiviral agent against hepatitis further described in U.S. Pat. No.5,928,638.

The stem cells are transplanted into the recipient subject. This isgenerally effected using methods well known in the art, and usuallyinvolves injecting or introducing the treated stem cells into thesubject using clinical tools well known by those skilled in the art(U.S. Pat. Nos. 6,447,765, 6,383,481, 6,143,292, and 6,326,198).

For example, introduction of the stem cells of the present invention canbe effected via intravascular administration, including intravenous orintraarterial administration, intraperitoneal administration, and thelike. Cells can be injected into a mol Fenwall infusion bag usingsterile syringes or other sterile transfer mechanisms. The cells canthen be immediately infused via IV administration over a period of time,such as 15 minutes, into a free flow IV line into the patient. In someembodiments, additional reagents such as buffers or salts may be addedas well. The composition for administration must be formulated, producedand stored according to standard methods complying with proper sterilityand stability.

Stem cell dosages can be determined according to the prescribed use. Ingeneral, in the case of parenteral administration, it is customary toadminister from about 0.01 to about 5 million cells per kilogram ofrecipient body weight. The number of cells used will depend on theweight and condition of the recipient, the number of or frequency ofadministrations, and other variables known to those of skill in the art.

After administering the cells into the subject, the effect of thetreatment may be evaluated, if desired, as known in the art. Thetreatment may be repeated as needed or required.

The invention also provides a pharmaceutical composition comprising atherapeutically effective amount of an HGF, or an active portion thereofto be administrated alone or co-administrated with SCF. Morespecifically the invention provides pharmaceutical compositions fortreating a disorder requiring cell or tissue replacement.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “mnobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Increased HGF Mediated Motility and CXCR4 Mediated Migrationof Human CD34⁺ Progenitors to an Injured Liver

It is well known that HGF is upregulated in the injured liver [Armbrust(2002) Liver 22:486-494) and that addition of human HGF increases thelevels of human albumin producing cells in CCl₄ injured-livers inengrafted immune deficient murine chimeras [Wang (2003) Blood (epubahead of print)]. Therefore, it was hypothesized that HGF may alsoparticipate in the regulation of human CD34⁺ cell migration andrecruitment to the injured liver.

Human cells—CB and adult mobilized peripheral blood (MPB) were obtainedfollowing informed consent and in accordance with procedures approved bythe human ethics committee of the Weizinann Institute. CD34⁺ cellenrichment was effected using magnetic bead separation as previouslydescribed [Kollet (2001) Blood 97:3283-3291]. CXCR4 expression wasdetermined by flow cytometry using purified anti human CXCR4 (clone12G5, R&D, Minneapolis, Minn.) and secondary F(ab′)2 fragment of goatanti mouse IgG FITC (Jackson, West Grove, Pa.).

Liver injury—Mice were injected intra peritoneously (IP) with 10, 15 or30 μl/mouse of CCl4 and liver samples were collected within a few hours,or 1-2 days following injection, as indicated.

Immunocytochemsity—CB CD34⁺ enriched cells were incubated for 40 hoursin RPMI supplemented with 10% FCS in the absence of cytokines, or in thepresence of SCF (50 ng/ml, R&D), HGF (100 ng/ml, PeproTech) or bothcytolines. Cells were plated on fibronectin (10 μg/cm², Calbiochem, SanDiego, Calif.) coated glass cover slips (2 hours, 37° C., 5% CO2), fixedfor 25 minutes in 3% paraformaldehyde, permeabilized for 5 minutes in0.5% TritonX100, both in PBS. Cells were indirectly immunolabeled withrabbit anti human CXCR4 polyclonal Ab (Chemicon, Temecula, Calif.),washed extensively with PBS and incubated with Phalloidin-TRITC (Sigma,Rehovot, Ill.) and goat anti rabbit Alexa 488 (Molecular Probes, Eugene,Oreg.). Following extensive washing with PBS, samples were mounted inElvanol (Mowiol 4-88; Hoechst, Frankfurt, Germany); All procedures werecarried out in a humidified atmosphere, at room temperature.Immunofluorescence was viewed and analyzed using a BioRad confocalmicroscope, at 100× magnification.

Chemotaxis—Migration of enriched CD34⁺ cells towards a gradient of SDF-1was determined by transwell assay as described [Peled (1999) Science283:845-848]. CD34⁺ cells were incubated for 40 hours in RPMIsupplemented with 10% FCS in the absence of human cytokines, or with SCF(50 ng/ml, 614 McKinley Place NE Minneapolis, Minn.), HGF (100 ng/ml,PeproTech, Rocky Hill, N.J.) or both cytokines prior to migrationtowards 125 ng/ml of SDF-1.

Results

Enriched CB CD34⁺ cells were cultured for 40 hours in the absence ofcytokines, or in the presence of stem cell factor, which is known toinduce CXCR4 expression and SDF-1 dependent migration [Peled (1999)Science 283:845-848], HGF or a combination of both cytokines. Cells werethen incubated with anti CXCR4 and/or anti-polymerized actin, washedextensively with PBS and incubated with Phalloidin-TRITC and goat antirabbit Alexa 488. While CD34⁺ cells cultured in the absence of cytokinesmaintained a round shape, cells cultured with SCF were spread andpolarized. Interestingly, HGF alone induced formation of actin-basedprotrusions from the cell surface and the combination of SCF and HGFpromoted lamellipodia formation, a phenotype distinct from that observedwith SCF or HGF alone (data not shown). Most importantly, thesecytoskeletal rearrangements were associated with CXCR4 upregulation(FIG. 1 a) and a functionally enhanced chemotactic response to SDF-1(FIG. 1 b). HGF did not induce chemotaxis of human progenitors alone(data not shown). However, HGF increased the motility of humanprogenitors and synergized with SCF to potentiate both CXCR4 expressionand SDF-1-induced directional migration.

These unexpected findings suggest an important role for HGF infacilitating motility and directional migration of human CD34⁺ cells inresponse to injured liver stress signals.

Increased expression of HGF MRNA in the BM and liver of irradiated mice.

Example 2 Irradiation Upregulates of HGF in the Liver and in the BoneMarrow

It was explored whether irradiation, which is used prior totransplantation and is known to upregulate a variety of cytokines suchas SDF-1, induces upregulation of HGF in the liver and in the bonemarrow.

NOD/SCID mice were sublethally irradiated (375 cGy). Liver and BM samplecollected 24 and 48 hours later, together with a sample from anon-irradiated mouse, were homogenized in Tryreagent (MRC). The MRNA wasextracted and subjected to RT-PCR with specific primers. FIG. 2 showsthat expression of HGF is increased 1 irradiation in both, the BM and inthe liver.

Example 3 HGF Synergies with SCF to Increase the Repopulating Potentialof CB CD34⁺ Cells

As demonstrated in example 1, HGF increases the motility of humanprogenitors and synergies with SCF to potentiate both, CXCR4 expressionand SDF-1-induced directional migration.

Further experiments were carried out to explore whether HGF synergieswith SCF to increase the repopulating potential of hematopoieticprecursors. Thus, human CB CD34+ cells were cultured in the presence ofSCF, HGF or the combination of both. NOD/SCID mice were irradiated (375cGy) and transplanted with the cultured cells. One month later, BM oftransplanted mice was examined for the level of human engrafting cells.

CB CD34+ cells were cultured in the presence of SCF (50 ng/ml), HGF (100ng/ml) or both, in 10% FCS+RPMI, for 36 h. NOD/SCID mice (3 per group)were irradiated (375 cGy) and transplanted 24 h later with culturedcells derived from 2×105 cells/mouse. One month later, BM oftransplanted mice was examined for the level of human engrafting cells,by using anti human CD45-FITC Ab and FACSCalibur.

TABLE 2 Human cell Treatment engraftment (%) Control 16.9 10.2 2.6 SCF23.6 19.3 1.5 HGF 7.9 7.4 16.9 SCF + HGF 7.0 33.7 21.2

The results in Table 2 show that cells treated with HGF alone repopulateBM similarly to the control cells and at a significant lesser extentthan cells pretreated with SCF. In contrast cells co-stimulated with SCFand HGF show increased repopulation capability in comparison to celltreated with HGF or SCF alone.

Example 4 HGF Increases the Potential of BM Cells to Migrate TowardsSDF-1 and the Rate of Progenitor Cell Mobilization, Following CCl4Injury

As demonstrated in example 1, ex-vivo HGF administration to progenitorstem cells induces CXCR4 expression on the cell surface and enhancemigration of the cells to SDF-1.

The following experiments were carried out to find the effect of in vivoadministration of HGF in repopulation following organ injury.

For that purpose, NOD/SCID mice were treated with CCl4 alone (to induceinjury), with the combination of CCl4 followed by HGF or remaineduntreated (control). The mice were sacrificed, the bone marrow cellsextracted, and migration of such BM cells to SDF-1 was monitoredin-vitro (FIG. 3).

NOD/SCID mice were treated with a single injection of CCl4 alone (10 mlCCl4), or with CCl4 followed by 4 consecutive daily injections of HGF(1.5 mg/mouse, CCl4+HGF) starting 2 days later. SDF-1-induced migrationby BM cells derived from these treated mice were compared toSDF-1-induced migration by BM cells derived from non treated mice (ctrl)or from mice treated with G-SCF (300 mg/Kg, 5 consecutive days).

BM cells isolated from CCl4 treated mice showed similar level of SDF-1dependent migration as the level of migration obtained with BM cellsfrom control non treated mice. In contrast, BM cells isolated from G-SCFtreated mice showed enhanced level of SDF-1 dependent migration incomparison to the level of migration observed with BM from control nontreated mice. Unexpectedly, BM cells isolated from mice receiving thecombined treatment with HGF and CCl4 showed enhanced level of SDF-1dependent migration in comparison to the level of migration observedwith BM cells from CCl4 treated or non-treated mice.

In light of the increase in the migration capability of BM cellsobserved in HGF/CCl4 treated mice, it was hypothesized that HGF/CCl4treated mice may exhibit also an increase in the level of precursorprogenitor cell in the blood circulation, consequently to BMmobilization.

To explore this hypothesis, the number of progenitor precursors in thecirculation was monitored in CCl4 treated, in CCl4 and HGF treatedversus non-treated mice (FIG. 3).

The results obtained show that induction of injury or inflammation, byCCl4 treatment, increased the number of progenitor precursors in theblood circulation. This results suggests that induction of injury orinflammation by CCl4 facilitates mobilization of progenitor precursorsfrom the bone marrow to the blood but at a lesser extent than thepositive control for mobilization (see G-SCF). However, inflammation orinjury induced by CCl4 treatment in combination with HGF administration,induced a further increase in the level of progenitor precursors in theblood circulation.

The results obtained suggest administration of HGF to a subjectsuffering of organ inflammation and/or injury, to facilitate selfrepopulation and/or self engraftment to the injured organ, due toincreased progenitor levels in the cell blood circulation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. A method of generating stem cells suitable for transplantation, themethod comprising: (a) collecting stem cells expressing c-met; exposingsaid stem cells to hepatocyte growth factor (HGF) or an active portionthereof alone; and (c) isolating stem cells having CXCR4 levels above apredetermined threshold, to thereby generate stem cells suitable fortransplantation.
 2. The method of claim 1, wherein collecting said stemcells is effected by: (i) a stem cell mobilization procedure; and/or(ii) a surgical procedure.
 3. The method of claim 1, wherein said stemcells are hematopoietic stem cells.
 4. The method of claim 1, whereinsaid hematopoietic stem cells are CD34⁺ hematopoietic stem cells.
 5. Themethod of claim 4, wherein said hematopoietic stem cells areCD34⁺/CD38^(−/low) hematopoietic stem cells.
 6. The method of claim 1,wherein said stem cells are mesenchymal stem cells.
 7. The method ofclaim 1, wherein said exposing said stem cells to said HGF or saidactive portion thereof, is effected by: (i) expressing a polynucleotideencoding said HGF or an active portion thereof in said stem cells; andor (ii) contacting said stem cells with said HGF or an active portionthereof.
 8. The method of claim 1, wherein said isolating stem cellshaving CXCR4 levels above said predetermined threshold is effected byFACS.
 9. The method of claim 8, further comprising determining homingcapabilities of said stem cells having CXCR4 levels above saidpredetermined threshold following step (c).